Inteins (internal protein fragments) are in-frame intervening sequences that disrupt the coding region of a host gene. These internal protein elements mediate the post-translational protein splicing process, catalyzing a series of reactions to remove the intein from the protein precursor and to ligate the flanking external protein fragments, known as exteins, into a mature protein (Perler, F. B. Cell 92:1-4 (1998)). A typical intein element consists of 400 to 500 amino acid residues and contains four conserved protein splicing motifs, although mini-inteins approximately 150 amino acids in size have been identified (Perler, F. B. Nucl. Acids. Res. 28:344-345 (2000)). Over all, nearly 140 putative inteins have been found from prokaryotes (archaea and eubacteria) and single cell eukaryotes such as algae and yeast, mostly through genome sequencing projects (Perler, F. B. (2000), supra). The majority of these inteins mediate maturation of enzymes involved in replication, DNA repair, transcription, or translation. Protein splicing has yet to be observed in a multicellular organism.
Since the discovery of inteins, much has been done to elucidate their functional mechanisms and potential applications. The complete splicing mechanism, consisting of four coupled nucleophilic displacements between three conserved amino acid residues at intein-extein junctions, is reviewed by Noren, C. J. et al. (Angew. Chem. Int. Ed. 39:450-466 (2000)). This protein splicing mechanism has been reconstituted in vivo and in vitro, demonstrating that inteins could be used as powerful tools for protein modification and engineering (Perler, F. B. and Adam, E. Curr. Opin. Biol. 11:377-383 (2000)). Additionally, both trans-splicing and cis-splicing have been studied.
Protein trans-splicing is a reaction that ligates separate proteins into a hybrid molecule, mediated by a pair of split inteins. Therefore, protein trans-splicing offers great advantages over cis-splicing. For example, trans-splicing can permit the synthesis of highly toxic proteins, when a strategy is applied such that single cells only contain a portion of the toxic protein, while the entire toxic protein is synthesized in vitro. Additionally, it may permit expression of a gene from two different loci of a genome or two cellular compartments. To study protein trans-splicing, artificial split inteins have been generated, in which the N-terminal half intein (Int-n) usually contains the critical A and B splicing motifs and the C-terminal half intein (Int-c) contains the C and F motifs. When the half inteins are fused, each half intein being associated with a partial protein, the two partial proteins can be spliced to form a hybrid product both in vitro and in vivo (Mills, K. V. Proc. Nat. Acad. Sci. USA. 95: 3543-3548 (1998); Southworth, M. W. et al. EMBO. 17:918-926 (1998); Wu, H. et al. Biochimica et Biophysica Acta 187:422-432 (1998); Yamazaki, T. et al. J. Am. Chem. Soc. 120:5591-5592 (1998)). The general utility of these artificial inteins, however, is hindered by a strict requirement for urea treatment to denature and renature the proteins.
The Ssp DnaE inteins are the only known natural split inteins. This intein class was identified from the split DnaE genes of Synechocystis sp. PCC6803, which encode the catalytic subunit α of DNA polymerase III (Wu, H. et al. Proc. Natl. Acad. Sci. USA. 95:9226-9231 (1998)). The N-terminal half of the DnaE protein containing 774 amino acid residues is fused to the N-terminal 123 amino acid Ssp DnaE intein sequence. The remaining 36 amino acid residues of the C-terminal half of the Ssp DnaE intein are fused separately to the C-terminal half of the DnaE protein, containing 423 amino acids. These two genes are located 745 kB apart on opposite strands of the Ssp PCC6803 genome, although their protein product is an intact catalytic subunit of 1197 amino acid residues lacking any intein sequence due to the intein-mediated protein trans-splicing. In general, efficiency of the protein trans-splicing is usually higher when using Ssp DnaE natural split inteins instead of artificial split inteins (Martin, D. D. et al. Biochemistry. 40:1393-1402 (2001)).
The split Ssp DnaE inteins are also unique in their ability to catalyze the trans-splicing reaction even when two halves of the exteins are foreign proteins. For example, using two compatible plasmids each with an unlinked gene fragment, E. coli was found to be able to: (1) express the two gene fragments containing halves of a herbicide-resistant form of bacterial acetolactate synthase II (ALAS II) gene each fused to the split intein sequences; and (2) form a herbicide-insensitive enzyme in vivo (Sun, L. et al. Appl. Envir. Micro. 67:1025-1029 (2001)). When a wild type corn ALS gene was similarly used, the expected size of the reconstituted enzyme was formed in vivo (in E. coli) but no evidence was presented as to whether it was functional or whether intein-mediated splicing can occur in plant cells. A similar study was performed, again in E. coli, whereby it was determined that an artificially split bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene could be reassembled as a functional enzyme via intein trans-splicing (Chen et al. Gene 263:39-48 (2001)).
In both Sun et al., supra, and Chen et al., supra, it is suggested that the split Ssp DnaE inteins may be used in agriculture where plants may be genetically modified by utilizing trans-splicing technology to contain herbicide resistant transgenes in crops. In theory, this could be accomplished by expressing inactive gene fragments in separate DNA locations, and only allowing protein activity to be generated following trans-splicing. However, the art is silent concerning the methodology that would be necessary for one skilled in the art of plant transgene expression to practice this concept, and no demonstration of the intein-mediated protein splicing technique exists in eukaryotes. Further, there has been no demonstration that inteins are able to function in higher organisms, such as plants.
The advent of genetically modified crops holds the promise of improving crop yield and quality. These benefits are conferred via the transformation of crop plants with new transgenes encoding desirable traits. Plants are increasingly being looked to as platforms for the production of materials, foreign to plant systems. As the art of genetic engineering advances, it will be possible to engineer plants for the production of a multiplicity of monomers and polymers, currently only available by chemical synthetic means. The accumulation of these materials in various plant tissues will be toxic at some level and it will be useful to tightly regulate the relevant genes to prevent expression in inappropriate plant tissues.
Currently, few methods exist that provide for tightly regulated transgene expression. Non-specific expression of transgenes in non-target cells, tissues, or generations hinders plant transgenic work. This is important where the goal is to produce such high levels of materials in transgenic plants that may be phytotoxic or adversely affect normal plant development. Conditional transgene expression would enable economic production of desired chemicals, monomers, and polymers at levels likely to be phytotoxic to growing plants by restricting their production to transgenic crop biomass (production tissue) either just prior to or after its harvest for extracting the desired product. Therefore, the development of transgene expression in plants is limited both by the lack of a commercially usable conditional expression system and the difficulty in attaining reliable, high-level expression.
Transgenic trait expression often requires temporal and tissue-specific control of a transgene. Thus, binary expression systems are utilized such that traits are not expressed in the hybrid parents but are expressed in F1 hybrid progeny of parents each carrying an element of the binary system. A good example of this binary system is site-specific recombination (SSR), where one parent carries a site-specific recombinase and the other carries the inactive trait. The expression of this trait gene (TG) is blocked by the presence of a ‘blocking’ or ‘STOP’ fragment, flanked by the cognate SSR sites, which blocks transcription and/or translation of the TG (Yadav et al., WO 01/36595 A2; WO 00/17365 A2; U.S. Pat. No. 6,077,992).
Recombinase expression in the progeny leads to SSR and removal of the ‘blocking’ DNA fragment, thereby permitting transgene activation. Similarly, when generational control of removal of a TG flanked by SSR sites is required, one can cross one line carrying the TG and another expressing the recombinase gene.
Site-specific recombination [Odell et al., Plant Physiol. 106:447-458 (1994); Odell et al., PCT Int. Appl. WO 9109957 (1991); Surin et al., PCT Int. Appl WO 9737012 (1997); Ow et al., PCT Int. Appl. WO 9301283 A1 (1992); Russel et al., Mol. Gen. Genet. 234:49-59 (1992); and Hodges et al. (U.S. Pat. No. 6,110,736)] in plants has been demonstrated. Furthermore, regulated SSR in plants and the use of mutant sites to enhance the specificity of Cre-mediated recombination in conjunction with chimeric Cre genes under the control of available regulated promoters has also been demonstrated in plants (Yadav et al., WO 01/36595 A2; WO 00/17365 A2; EP1115870 A2). Further, directed excision of a transgene from the plant genome has been reported using recombinase specific-sites and a recombinase (Russel et al., Mol. Gen. Genet. 234:49-59 (1992); Ow et al., PCT Int. Appl. WO 9301283 A1 (1992)).
One limitation of the above-mentioned approach, however, is that only one parent can carry the recombinase and the other its substrate containing the cognate SSR sites. As a result, the trait locus is heterologous in F1 hybrid progeny. Another limitation of the existing SSR techniques is that the site-specific recombinase may show toxicity through chromosomal rearrangements in plants and animals. For example, the Cre transgene under some plant promoters (e.g., the Bcp 1 gene) show Cre phytotoxicity in some transformants, even when they have the required regulation specificity. This results in pollen sterility with Bcp1:Cre and unwanted spread of active Cre recombinase in future generations. Such toxicity has also been reported in animal cells (see Schmidt et. al. PNAS, U.S.A. 97:13702-13707 (2000)). One solution to such toxicity, when the threshold of recombinase concentration for toxicity is higher than for recombination, is to contain the recombinase within the floxed DNA element such that upon SSR the recombinase gene is autoexcised; and thus, build up of recombinase is prevented. However, maintainence of these lines will be unlikely when the recombinase is under the control of a developmentally regulated promoter.
The problem to be solved therefore is to develop a system for conditionally regulating transgene expression through the implementation of site-specific recombinase systems such that the potential toxicity of the recombinase is minimized.
Applicant has solved the stated problem in the present invention through the development of a site-specific recombinase system based on intein-mediated protein splicing, comprising an inactive recombinase element and a trait expression construct containing an expressible transgene. The organization of the inactive recombinase element results in the splitting the site-specific recombinase into two inactive components that are unable to catalyze the SSR when present individually. However, when both inactive split recombinases are brought together, say, by a cross, the recombinase activity is restored through a split intein-mediated trans-protein splicing reaction. Once restored, the recombinase may act on the trait expression construct to regulate transgene expression.